title: Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons.
abstract: It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Sherry
authors.initials: S
authors.lastname: Dadsetan
authors.forename: Lasse K
authors.initials: LK
authors.lastname: Bak
authors.forename: Michael
authors.initials: M
authors.lastname: Sørensen
authors.forename: Susanne
authors.initials: S
authors.lastname: Keiding
authors.forename: Hendrik
authors.initials: H
authors.lastname: Vilstrup
authors.forename: Peter
authors.initials: P
authors.lastname: Ott
authors.forename: Renata
authors.initials: R
authors.lastname: Leke
authors.forename: Arne
authors.initials: A
authors.lastname: Schousboe
authors.forename: Helle S
authors.initials: HS
authors.lastname: Waagepetersen
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000410:Alanine Transaminase / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018920:Coculture Techniques; D005969:Glutamate Dehydrogenase / Q000502:physiology*; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005973:Glutamine / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D022124:Hyperammonemia / Q000139:chemically induced / Q000378:metabolism; D008717:Methionine Sulfoximine / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
chemical_list: D005973:Glutamine; D008717:Methionine Sulfoximine; D000641:Ammonia; D005969:Glutamate Dehydrogenase; D000410:Alanine Transaminase; D005974:Glutamate-Ammonia Ligase; D000409:Alanine